The present invention discloses methods for the identification of Fritillaria species.
Beimu, bulbs of the plant genus Fritillaria, is an important traditional Chinese herbal drug commonly used as an antitussive and expectorant (Li, P. et al., 1993, J. China Pharm. Univ. 24: 360-362). At least 25 species and varieties of Fritillaria are described as Beimu on commercial markets (Li, P. and Xu, G. J., 1993, J. Plant Res. Envir. 2: 12-17; Li, P. et al., 1990a, Chin. Trad. Herbal Drugs 21: 26-29; Li., P. et al., 1990b, J. China Pharm. Univ. 21: 19-25). However, these species and varieties differ extensively in terms of their medical efficacy, general abundance, price and toxicity (Liu, G. et al., 1996, Pharm. Res. 13: 10), and it is important for both producers, consumers and regulators to be able to identify the origin of a particular plant.
Current approaches to herbal identification depend on morphological, anatomical and chemical analyses, but these characteristics are often affected by environmental and/or developmental factors during plant development (Li, P. et al., 1994, J. Plant Res. Envir. 3: 60-63; Li, P. et al., 1991, Acta Bot. Yunnanica 13: 41-46). Many of the Fritillaria species have bulbs which are very difficult to differentiate by anatomical and morphological characteristics. Furthermore, chemotaxonomical studies can be hampered by the crude processing techniques often employed in the preparation of herbal medicines.
Work disclosed here shows for the first time that the 5S-ribosomal RNA (5S-rRNA) gene sequence contains spacer regions (introns) which are polymorphic for Fritillaria species. In all higher eukaryotes coding regions of the 5S-rRNA gene are separated from each other by simple spacers. The gene occurs as a tandem repeated unit (cistron) consisting of a xc2x1120 bp coding region separated by a 300 bp spacer region (Dovorak, J. et al., 1989, Genome 32: 1003-1016). The spacer regions are valuable in different species. Experiments (below) show that informative DNA sequence differences exist in the sixteen Fritillaria species and varieties examined (SEQ ID NOs: 1-16, FIGS. 5A through 5E). 5S-rRNA DNA sequences have been used to rapidly differentiate between Fritillaria species using a variety of techniques which enable comparison between the DNA sequence.
Plant phylogenisation is known in the art (see for example GB 2310718, U.S. Pat. No. 5,849,492, U.S. Pat. No. 5,738,988, U.S Pat. No. 5,723,507) but it has not been previously suggested that it may be achieved by the method of the present invention.
Thus according to the present invention there is provided a method for determining the phylogeny of sample Fritillaria genetic material, comprising the steps of:
i) characterising the spacer region of the 5S-rRNA gene of said sample genetic material;
ii) comparing the characterised sample genetic material of step (i) with a characterised 5S-rRNA gene spacer region of control Fritillaria genetic material having a known phylogeny; and
iii) correlating the results of comparison step (ii) to determine the phylogeny of the sample genetic material.
The method of the present invention may be used to determine the exact phylogeny of sample genetic material, or it may alternatively be used to provide negative results i.e. determine what the sample genetic material phylogeny is not. The method may also be used to determine the quantity of sample Fritillaria genetic material. Thus the method may be used to determine the presence and quantity of specific Fritillaria genetic material in a sample.
As well as being applicable to determining the phylogeny of sample Fritillaria genetic material (e.g. determine its species and variety), the method of the present invention may also be applied to other genetic material, particularly other plant genetic material. Thus as well as determining the species of genetic material, it may be used to determine the phylogeny of genetic material (e.g. determine its genus, family etc.). Naturally, in such a method to determine the phylogeny of non-Fritillaria genetic material, the control genetic material used would have to be non-Fritillaria.
The spacer region may be characterised by any desired technology, for example by sequencing, restriction fragment length polymorphisms (RFLP), polymerase chain reaction (PCR) (for example using discriminatory primers), 5S-rRNA gene spacer region length polymorphism and single strand conformational polymorphisms (SSCP). Discriminatory oligonucleotide primers may also be employed in PCR reactions according to the present invention. By using PCR technology, nanogram quantities of DNA are required to amplify and yield sufficient amount of template DNA for molecular genetic analysis.
Alternatively, characterisation may be achieved by studying the said (sample or control) hybridisation of a single strand of genetic material to a complementary strand of genetic material having known characteristics. This complementary strand of genetic material may include a known sequence or sequences or have a sequence or sequences which are specifically phylogeny-related, or it may simply be of a known phylogeny, i.e. the sequence need not be known. The complementary genetic material may be attached to a solid support in order to aid in the characterisation process. For example, hybridisation to a complementary strand at a specific position in an array of complementary strands may be assayed. The characterisation may be achieved using xe2x80x9cgene chipxe2x80x9d technology (Palecek, 1988, Bioelectrochemistry and Bioenergetics, 20: 179-199; Schena, M. et al., 1995, Science, 270: 467-470; Pease, A. C. et al., 1994, PNAS USA, 91: 5022-5026).
The DNA manipulation techniques employed in the present invention include those known to a person skilled in the art of characterising and differentiating between known and unknown genetic materials, for example: PCR (McPherson, M. J. et al., 1991, PCR: A practical approach, Oxford University Press, Oxford); DNA cloning and Southern hybridisation (Sambrook, J. et al., 1989, Molecular cloning: a laboratory manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y.); restriction fragment length polymorphism (RFLP) (Davies, K. E., 1988, Genome analysis: a practical approach, IRL Press, Oxford); single strand conformational polymorphism (SSCP) (Orita, M. et al., 1989, PNAS USA 86 (8): 2766-2770); and sequencing (Sanger, F. et al., 1977, PNAS USA 74(12): 5463-5467).
The control genetic material used in any method according to the present invention may have the sequence of any one of the group comprising the introns of SEQ ID NOs: 1-16. These sequences include entire introns and it will be readily apparent to on skilled in the art that distinctive fragments (portions) of the introns may be used, i.e. fragments not shared by (common to) other known 5S-rRNA gene introns. Such distinctive fragments may be insufficiently complementary to a 5S-rRNA spacer region of a non-control organism to prevent specific detection of said nucleic acid molecule hybridized with said control 5S-rRNA nucleic acid under stringent hybridization conditions.
The sample genetic material may comprise genomic DNA. Genomic DNA may be isolated from Fritillaria species and varieties using standard techniques (see for example Gelvin, S. B. et al., 1993, Plant molecular biology manual, Kluwer Academic, Dordrecht).
Also provided according to the present invention is a nucleic acid probe for determining the phylogeny of sample Fritillaria genetic material comprising a nucleic acid selected from any one of the group of the introns of SEQ ID NOs: 1-16 or a distinctive fragment thereof. The nucleic acid probe may be free in solution for specifically detecting the presence or amount of a target organism or group of organisms containing ribosomal nucleic acid, in a sample which may include non-target organisms containing ribosomal nucleic acid.
Also provided according to the present invention is the use of a sequence selected from any one of the group consisting the introns of SEQ ID NOs: 1-16 or a distinctive fragment thereof in the manufacture of a kit for determining phylogeny of sample Fritillaria genetic material.
Also provided according to the present invention is a test kit for determining the phylogeny of sample Fritillaria genetic material characterised in that it comprises at least one sequence selected from the group consisting the introns of SEQ ID Nos: 1-16 or a distinctive fragment thereof.
Experiments (below) have shown that not only is there polymorphism in the 5S-rRNA gene spacer region at an intra-species level, but also at the intra-variety level. Thus the method of the present invention may determine the species or variety of sample genetic material obtained from the genus Fritillaria.